113 Effect of isospintanol and sericin supplementation in IVM medium of bovine in vitro embryo production and its cryotolerance after vitrification

In vitro embryo production (IVEP) is an assisted reproductive technology (ART) that has a notorious impact on the productive efficiency of herds, obtaining a high number of embryos with desired genetics. However, there are limitations to this technology, such as the low cryotolerance of embryos due to the accumulation of lipids in the cytoplasm induced by the addition of fetal bovine serum (FBS) in the medium during IVEP. This study evaluates the supplementation of sericin and isospintanol, a natural polymer produced by the silkworm (Bombyx mori) and a natural antioxidant produced by espintana plant (Oxandra xylopioides), as an alternative protein source to FBS in in vitro maturation (IVM) medium. Slaughterhouse-derived bovine oocytes (n = 2260) were in vitro matured for 22–24 h in tissue culture medium 199, supplemented with 0.2 mM C3H3NaO3, 10 ug/mL FSH, 100 uM cysteamine, 0.5% ATB, and four different concentrations of sericin (S1: 0.1%, S2: 0.5%, S3: 1%, S4: 2.5%) and three concentrations of isospintanol (10 uM, 20 uM, 30 uM) at 38.5°C in 6.5% CO₂ humidified air. 10% of FBS was used as a control (CG). Additionally, one concentration of sericin, isospintanol, and the combination of both was selected to be used in IVM medium and evaluated its effect on embryo development and cryotolerance. The maturation rate (MR) was evaluated by extrusion of the first polar body. For IVF, in vitro-matured oocytes were co-incubated with 2 × 10⁶ spermatozoa per mL in an atmosphere of 21% O₂ at 38.5°C in 6.5% CO₂ humidified air for 18–20 h. Presumptive zygotes were cultured in vitro in 50-μL drops of synthetic oviducal fluid (SOF) medium with 5% FBS on 6.5% CO₂ in air at 38.5°C. Cleavage was determined 72 h post-fertilisation and blastocyst stage was evaluated at Day 7. Expanded blastocysts at Day 7 were vitrified and embryo re-expansion was evaluated 24–72 h post-warming according to Kuwayama et al. (2005). Each experiment was repeated three times and data were analysed by Fisher’s exact test (P < 0.05). No differences were observed between S1, S2, and S3 (S1: 110/171-64%, S2:119/179-66%, S3:105/189-56%); even so, S4 showed lower MR (98/215-46%) and CG showed higher (267/337-79%) MR. Moreover, no differences were observed between the concentration of isospintanol (10 uM: 133/161-83%, 20 uM: 117/153-76%, 30 uM: 126/167-75%) and CG (267/337-79%). 1% of sericin (S3), 20 uM of isospintanol, and the combination of both (S3 + 20 uM) were selected for supplementation in IVM medium. We found statistical differences in cleavage rate between S3 (91/151-60%) and the other groups (20 uM: 114/160-71%, S3 + 20 uM: 149/211-71%, and CG: 115/166-69%). Supplementation with 20 uM of isospintanol and CG showed higher blastocyst rate compared with the other groups (20 uM: 58/168-34%, CG: 58/166-35% vs S3: 37/151-24% and S3 + 20 uM: 41/211-19%). Differences were found in re-expansion after warming with S3 + 20 uM and CG compared with the other groups (S3 + 20 uM: 8/31-26%, CG: 9/32-28% vs 20 uM: 21/33-64% and S3: 13/20-65%). Our results suggest that sericin is a viable alternative to replace FBS in IVM medium and 20 uM of isospintanol increases the blastocyst rate similar to CG. However, the combination of both does not favour the maturation, development, and cryotolerance of bovine embryos produced in vitro.